Preparation of Nuclear Extracts from HeLa cells.

HeLa cells are the archetypal tissue culture cell line for the preparation of mammalian cell-free systems. These cells, derived from a cervical carcinoma, have been maintained in culture since the late 1940s. They grow with a doubling time of ∼24 h and can be cultured in medium containing fetal bovine serum (optimal serum for growth) or medium containing horse serum (in which they grow more slowly, but this serum is considerably less expensive). Nuclear extracts prepared from these cells have been used to determine the mechanisms of splicing and polyadenylation, and such extracts have been characterized extensively. HeLa cells are usually the cell type of choice for initiating cell-free analysis of nearly any aspect of mammalian gene expression. In some instances (e.g., analysis of tissue-specific alternative splicing), it is necessary to use nuclei from a different cell type. We have found that the protocol described here can be used successfully to prepare active nuclear extracts from a wide variety of tissue culture cells, including Drosophila S2 cells.